Вы находитесь на странице: 1из 246

Ghasem Hosseini Salekdeh

Editor

Ghasem Hosseini Salekdeh Editor Agricultural Proteomics Volume 1 Crops, Horticulture, Farm Animals, Food, Insect and

Agricultural Proteomics Volume 1

Crops, Horticulture, Farm Animals, Food, Insect and Microorganisms

Salekdeh Editor Agricultural Proteomics Volume 1 Crops, Horticulture, Farm Animals, Food, Insect and Microorganisms

Agricultural Proteomics Volume 1

Ghasem Hosseini Salekdeh

Editor

Agricultural Proteomics Volume 1

Crops, Horticulture, Farm Animals, Food, Insect and Microorganisms

Editor Ghasem Hosseini Salekdeh Department of Systems Biology Agricultural Biotechnology Research Institute of Iran Karaj Iran

ISBN 978-3-319-43273-1 DOI 10.1007/978-3-319-43275-5

Library of Congress Control Number: 2016946323

ISBN 978-3-319-43275-5 (eBook)

© Springer International Publishing Switzerland 2016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microlms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specic statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.

Printed on acid-free paper

This Springer imprint is published by Springer Nature The registered company is Springer International Publishing AG Switzerland

Preface

Although the global food production has increased in recent decades, the global food demand increases more rapidly than production. It has been reported by FAO that demand for cereals will increase by 70 % by 2050 as an outcome of both larger populations and higher per-capita consumption among communities with growing incomes. To meet higher demand, growing more food at affordable prices becomes even more important. Agricultural proteomics can play a role in addressing the growing demand for food. The application of proteome science in agriculture has allowed researchers to identify a broad spectrum of proteins in living systems and associates them to many major traits. It may give clues not only about nutritional value, but also about yield production and food quality and how environments affect these factors. In recent years, technical improvements in the mass spectrometry, bioinformatics, protein extraction, and separation have made the high-throughput analysis of agricultural products feasible and the reproducibility of the technology has reduced errors in assaying protein levels. Meanwhile, the application of mass spectrometry-based quanti cation methods has become mainstream in recent year. The rapid advances of genome-sequencing tools also paved the way to sequence the full genome of many crops, animals, insects, and microorganisms. This provided Proteomics Scientist with a huge number of reference genome and genes for genome-wide proteome analysis. An emerging eld of the proteomics aimed to integrate knowledge from basic sciences to translate it into agricultural applications to solve issues related to eco- nomic values of farm animals, crops, food security, health, and energy sustain- ability. Given the wealth of information generated and to some extent applied in agriculture, there is a need for more efcient and broader channels to freely dis- seminate the information to the scienti c community. This book will cover several topics to elaborate how proteomics may enhance agricultural productivity. These include crop and food proteomics, farm animal

vi

Preface

proteomics, aquaculture, microorganisms, and insect proteomics. It will also cover several technical advances, which may address the current need for comprehensive proteome analysis.

Karaj, Iran

Ghasem Hosseini Salekdeh

Contents

1 Applications of Quantitative Proteomics in Plant Research Mehdi Mirzaei, Yunqi Wu, David Handler, Tim Maher, Dana Pascovici, Prathiba Ravishankar, Masoud Zabet Moghaddam, Paul A. Haynes, Ghasem Hosseini Salekdeh, Joel M. Chick and Robert D. Willows

 

1

2 Seed Proteomics: An Overview Kanika Narula, Arunima Sinha, Toshiba Haider, Niranjan Chakraborty and Subhra Chakraborty

 

31

 

53

 

. Javad Gharechahi, Mehrshad Zeinolabedini and Ghasem Hosseini Salekdeh

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

67

5 Holistic Sequencing: Moving Forward from Plant Microbial Proteomics to Metaproteomics Behnam Khatabi, Neda Maleki Tabrizi and Ghasem Hosseini Salekdeh

 

87

6 Proteomics in Energy Crops Shiva Bakhtiari, Meisam Tabatabaei and Yusuf Chisti

 

105

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

117

Chiew Foan Chin

 

127

9 Proteomic Applications for Farm Animal Management Ehsan Oskoueian, William Mullen and Amaya Albalat

 

147

viii

Contents

10 Applications of Proteomics in Aquaculture Pedro M. Rodrigues, Denise Schrama, Alexandre Campos, Hugo Os ó rio and Marisa Freitas

 

165

11 . Jeffrey E. Plowman and Santanu Deb-Choudhury

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

201

12 Proteomic Research on Honeybee Yue Hao and Jianke Li

 

215

Index .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

243

Contributors

Nagib Ahsan Division of Biology and Medicine, Brown University, Providence, RI, USA; Center for Cancer Research and Development, Proteomics Core Facility, Rhode Island Hospital, Providence, RI, USA

Amaya Albalat School of Natural Sciences, University of Stirling, Stirling, UK

Shiva Bakhtiari Biology Department, Concordia University, Montreal, Canada

Linda Bianco ENEA, Trisaia Research Center, Rotondella, MT, Italy

Alexandre Campos Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Porto, Portugal

Niranjan Chakraborty National Institute of Plant Genome Research, New Delhi, India

Subhra Chakraborty National Institute of Plant Genome Research, New Delhi, India

Joel M. Chick Department of Cell Biology, Harvard Medical School, Boston, MA, USA

Chiew Foan Chin Faculty of Science, School of Biosciences, University of Nottingham Malaysia Campus, Semenyih, Selangor Darul Ehsan, Malaysia

Yusuf Chisti School of Engineering, Massey University, Palmerston North, New Zealand

Santanu Deb-Choudhury Food & Bio-Based Products, AgResearch Lincoln Research Centre, Lincoln, New Zealand

Marisa Freitas Department of Environmental Health, Escola Superior de Tecnologia da Sa ú de do Porto, CISA/Research Center in Environment and Health, Polytechnic Institute of Porto, Gaia, Portugal

x

Contributors

Javad Gharechahi Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

Toshiba Haider National Institute of Plant Genome Research, New Delhi, India

David Handler Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia

Yue Hao Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing, China

Paul A. Haynes Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia

Behnam Khatabi Department of Biological Sciences, Delaware State University, Dover, Delaware, USA

Jianke Li Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing, China

Tim Maher Department of Biological Sciences, Macquarie University, Sydney, NSW, Australia

Mehdi Mirzaei Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia

Masoud Zabet Moghaddam Center for Biotechnology and Genomics, Texas Tech University, Lubbock, TX, USA

William Mullen Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK

Kanika Narula National Institute of Plant Genome Research, New Delhi, India

Ehsan Oskoueian Agricultural Biotechnology Research Institute of Iran (ABRII), East and North-East Branch, Agricultural Research, Education, and Extension Organization, Mashhad, Iran

Hugo Os ó rio Instituto de Investigaçã o e Inova çã o em Sa ú de - i3S (Institute for Research and Innovation in Health), University of Porto, Porto, Portugal; Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal; Faculty of Medicine of the University of Porto, Porto, Portugal

Dana Pascovici Australian Proteome Analysis Facility (APAF), Macquarie University, Sydney, NSW, Australia

Gaetano Perrotta ENEA, Trisaia Research Center, Rotondella, MT, Italy

Jeffrey E. Plowman Food & Bio-Based Products, AgResearch Lincoln Research Centre, Lincoln, New Zealand

Prathiba Ravishankar Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia

Contributors

xi

Pedro M. Rodrigues Departamento de Qu ímica e Farm á cia, Universidade do Algarve, CCMar, Faro, Portugal

Ghasem Hosseini Salekdeh Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Agricultural Research, Education, and Extension Organization, Karaj, Iran

Arthur R. Salomon Center for Cancer Research and Development, Proteomics Core Facility, Rhode Island Hospital, Providence, RI, USA; Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, USA

Denise Schrama Departamento de Qu ímica e Farm á cia, Universidade do Algarve, CCMar, Faro, Portugal

Arunima Sinha National Institute of Plant Genome Research, New Delhi, India

Meisam Tabatabaei Microbial Biotechnology Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran; Biofuel Research Team (BRTeam), Karaj, Iran

Neda Maleki Tabrizi Department of Agronomy and Plant Breeding, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran; Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Agricultural Research, Education, and Extension Organization, Karaj, Iran

Robert D. Willows Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia

Yunqi Wu Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia

Mehrshad Zeinolabedini Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Agricultural Research, Education, and Extension Organization, Karaj, Iran

Chapter 1

Applications of Quantitative Proteomics in Plant Research

Mehdi Mirzaei, Yunqi Wu, David Handler, Tim Maher, Dana Pascovici, Prathiba Ravishankar, Masoud Zabet Moghaddam, Paul A. Haynes, Ghasem Hosseini Salekdeh, Joel M. Chick and Robert D. Willows

Abstract Over the past two decades, we witnessed signi cant technological advances in Proteomics. Methodology and instrumentation have developed remarkably and proteomics has become a priority eld of research in biology. Furthermore, analysis of the entire proteome of many organisms became possible due to complete genome sequencing. The advances in mass spectrometry instru- mentation and bioinformatics tools have advanced quantitative proteomics tech- niques, resulting in important contributions to the biological knowledge of plants. In this chapter, we highlight the recent applications of proteomics in plants, in both model and non-model species. We then discuss the pros and cons of the major

Mehdi Mirzaei and Yunqi Wu have equally contributed in this work.

M. Mirzaei Y. Wu D. Handler P. Ravishankar P.A. Haynes R.D. Willows (& )

Department of Chemistry and Biomolecular Sciences, Macquarie University,

Sydney, NSW, Australia e-mail: robert.willows@mq.edu.au

T. Maher Department of Biological Sciences, Macquarie University, Sydney, NSW, Australia

D. Pascovici

Australian Proteome Analysis Facility (APAF), Macquarie University,

Sydney, NSW, Australia

M.Z. Moghaddam Center for Biotechnology and Genomics, Texas Tech University, Lubbock, TX, USA

G.H. Salekdeh Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Agricultural Research, Education, and Extension Organization, Karaj, Iran

J.M. Chick Department of Cell Biology, Harvard Medical School, Boston, MA, USA

2

M. Mirzaei et al.

quantitative approaches implemented in plant studies. Next, we describe the most studied post-translational modi cations (PTMs) in plant research, and lastly, we review the challenges of bioinformatics data analysis in the plant proteomics eld.

Keywords Mass spectrometry Quantitative proteomics Post-translational modi cation

1.1 Introduction

Over the past 15 years, signi cant technological advances in methodology and instrumentation have developed proteomics into a powerful tool. For this reason proteomics is a priority eld of research in biology. These advances combined with breakthrough technologies in genomics have allowed complete sequencing of the genome of an organism. Analysis of the entire proteome of an organism became possible for the rst time [ 1 ] due to complete sequencing. These advances allowed the study of not only entire proteomes but also the changes which occur under various perturbations. These studies provide the means to understand protein regulation, function and protein interactions [ 2 ]. In particular, proteomics can now, for a speci c complement of proteins present in a biological system at any one time, set out to answer the questions of what? , how? , where? , and when? for those proteins in that system [ 3 ]. Plant sciences in particular have bene ted from proteomics technology by studying and identifying metabolic pathways and protein functions, and identifying protein-protein interactions within model and crop plant systems. A PubMed search covering the period between 2001 and 2016 using the search terms plant and proteomics revealed over 5500 research and review articles. One of the rst proteomics plant studies was published by Pfannschmidt et al. in 2000. In this study the authors used two-dimensional gel electrophoresis for the analysis of the chloroplast polymerase A from mustard ( Sinapis alba L.) [ 4 ]. Since then, the evolution of mass spectrometry instrumentation (MS), sample preparation protocols, and bioinformatics data analysis platforms have advanced quantitative proteomics techniques, resulting in important contributions to the biological knowledge of plants. Most of the plant proteomics papers published to date on model plants apply to the subgroups of descriptive, subcellular, and com- parative proteomics [ 5 ]. Of these sub-areas, comparative proteomics is the most concerned with quantitative analysis, as it aims to highlight quantitative differences in proteins expressed between different genotypes, organelles, cell types, devel- opmental stages and external conditions of an organism. Such studies have pro- vided valuable information on plant biological processes, although without further functional analysis the proteomics data remain mostly descriptive [ 6 ]. A typical plant proteomics experimental pipeline from recent literature starts with a com- parative proteomics analysis, followed by bioinformatic analyses of protein expression data.

1

Applications of Quantitative Proteomics in Plant Research

3

1.2 Proteomics of Model and Non-model Plant Species

Quantitative proteomics approaches have largely been dedicated to model plant species [ 3 ]. In proteomics, these are species with a completely sequenced and well annotated genome. This information is typically made available in comprehensive databases such as that of the National Centre for Biotechnology Information (NCBI), which greatly assists in correct protein identi cation. At present, a total of 165 land plant species meet the model plant criteria. However, compared to the 3002 eukaryotic and 67,762 prokaryotic genomes sequenced to date (NCBI-April 2016), plants are highly under-represented. This relative shortfall is largely the result of plant genomes being greater in size and complexity, which makes them more difcult to sequence and annotate compared to other organisms [ 7 ]. To date, the plant species chosen as models are either species with relatively small genomes, or crop species of great economic value. The main application of plant proteomics being to better understand how crop species regulate development and respond to environmental changes [ 3 , 6 ]. The rst plant species to be sequenced was the dicot Arabidopsis thaliana in 2000 [ 8 ], which was soon followed by the complete sequencing of the economically important monocot rice ( Oryza Sativa ) in 2002 [ 9 ]. To date, proteomics studies concerned with model plants have provided key insights into a variety of protein families in plant systems, and how they are regulated and modi ed [ 6 ]. However, model plants alone do not possess all the features and processes of interest to plant biology [ 7 ]. Thus, orphan species , those without complete sequencing or adequate annotation, need to be investigated. Compared to model species the lack of genomic resources has seen orphan species largely neglected by quantitative proteomic studies [ 10 ]. Whether a species becomes a model is a trade-off between economic importance and the size and difculty of sequencing its genome. Therefore, orphan plant species tend to be either not of great economic signi cance, or possess large, complicated genomes or both [ 11 ]. However, crop species often possess large genomes which is largely the reason that many remain as orphan species [ 11 ]. Most current MS-based proteomics methods rely on comprehensive sequence databases for identi cation and quanti- tation, thus the analysis of these orphan species poses speci c challenges. These challenges can be at least partially overcome through utilising the sequence data- bases available from closely related species, or using databases of expressed sequence tags (EST) instead [ 12 ]. ESTs constitute a random snapshot of the expressed portion of the genome of a biological system at a moment in time in response to certain conditions. EST sequencing is seen as the most economical method of obtaining gene sequence information for orphan species [ 13 ]. Once collected, these ESTs are then added to databases for future protein identication in these species. However, the quality of EST sequences can vary greatly, so they need to be used very carefully in making con dent protein identications [ 14 ]. For those orphan species that do not have a sufcient number of ESTs available, protein identication may instead be achieved by relying on the established sequence databases of closely related model species

4

M. Mirzaei et al.

[ 15 ]. This method relies on the fact that many of the same genes are conserved between closely related species [ 16 ]. However, this is a less accurate method, especially for quantitative approaches, as it is not possible to know how many peptide assignments are taken into account, this due to sequence variation between species. Moreover, homologous proteins in different species often possess different functions and as the species become more distantly related the number of sequence mismatches increases [ 17 ]. Nevertheless, relying on this principle of shared protein sequence identity between related species has enabled quantitative proteomics approaches to be applied to many orphan species. In the event that no ESTs and no closely related model species exist for a particular orphan species, the method of de novo peptide sequencing may be used [ 18 ]. This method derives the sequence of the peptide solely from the tandem mass spectra output with no need for an existing sequence database [ 19 ]. A common error in using this approach is wrongly identifying amino acids from mass spectra that share very similar masses. However, the recent development of very high resolution mass spectrometry instrumentation suitable for proteomics analysis, for example Orbitrap MS, has greatly reduced this sequence ambiguity, increasing the accuracy of peptide sequencing and providing more con dent protein identi ca- tions [ 3 ]. This is still not really applicable when it comes to large-scale or shotgun proteomics studies, but it has been applied successfully in more targeted approaches.

1.3 Quantitative Proteomics Approaches

Numerous quantitative proteomics techniques have been used in proteomic research; however, not all of these are suitable for plant samples. Quantitative plant proteomics studies pose a number of unique challenges compared to animal and bacterial samples, such as: complications in protein extraction due to interfering secondary metabolites; separation of low abundance proteins; presence of incred- ibly high abundance proteins such as Rubisco in green plant tissues; genome multiploidy; and, most importantly, the absence of well-annotated and completed genome sequences [ 14 ]. Quantitative proteomic methodologies have undergone a number of advances which assist in overcoming these experimental challenges associated with plants. Evidently, 2-DE has been the most popular technique in plant research until approximately 2010; however, there has been a linear decrease in recent years. The second most used technique (label-free) reached its peak in 2012, however it seems the technique has lost its popularity and levelling off in the past couple of years. On the other hand, an emerging technology (chemical isobaric labelling approaches) is following an upward trend and it is estimated to keep on the same trend at least for the next few years to come (Fig. 1.1 ). Advances in high mass accuracy mass spectrometers, as well as multiplexing power of chemical isobaric labelling application in analysing the proteome and PTMs, are the main

1

Applications of Quantitative Proteomics in Plant Research

5

of Quantitative Proteomics in Plant Research 5 Fig. 1.1 Over all distribution of quantitative proteomics

Fig. 1.1 Over all distribution of quantitative proteomics techniques used in plant proteomics studies over the period of 2000 2015

reasons behind the increasing attractiveness of chemical isobaric labeling approa- ches (Fig. 1.1 ). In this section we brie y discuss the major quantitative proteomics approaches used in plant research.

1.3.1 Multi-dimensional Protein and Peptide Separation Methods

The rst quantitative proteomics studies were performed by separating proteins via two dimensional gel electrophoresis (2-DE). In this approach, proteins are separated based on their isoelectric point (pI) in the rst dimension, then separated based on molecular weight in the second dimension. Identication of proteins from 2-DE gels is obtained by excising protein spots, digesting with trypsin, extracting pep- tides, and then analysing the resultant peptides by mass spectrometry. One of the major advantages of 2-DE-based proteomic methods [ 20 , 21 ] is that they provide a visual output for protein pro ling and comparative mapping of expressed proteins between biological samples. Applications of 2-DE have been reported extensively in various plant species, such as Arabidopsis [ 22 , 23 ], soybean [24 , 25 ], rice [ 26 28 ], wheat [ 29 , 30 ], and many others [ 31 35 ]. However, there are considerable drawbacks to this technique, including the fact that certain groups of proteins are poorly represented on 2D gels. Such poorly represented proteins include those with large molecular weights, highly basic proteins with high pI s, and hydrophobic

6

M. Mirzaei et al.

proteins such as membrane spanning proteins which suffer due to poor solubility in 2-DE sample buffer. In addition, the detection of low abundance proteins is limited by total protein loading. A problem particular to green plant vegetative tissue samples is the presence of Rubisco, which can represent up to 60 % of the soluble protein in green plant tissue. Rubisco quantities interfere with the detection of many lower abundance proteins. For these reasons, 2-DE techniques are estimated to be only capable of detecting up to about 30 % of all cellular proteins. Furthermore, membrane proteins are poorly represented which is an issue for plant cells which are packed by various specialized membranous structures [ 36 ]. One particular technical drawback of 2DE technology, the detection of lower abundance proteins, has been addressed by recent advancements of uorescent dyes like SYPRO Ruby. The main advantages of SYPRO Ruby protein gel stains are their linear quantitation range, which spans almost over three orders of magnitude [ 37 ]. Despite these problems, 2-DE still remains a suitable approach for identication and visualization of intact proteins [ 38 ]. In addition, 2-DE allows de novo sequencing of individual protein spots, which facilitates identi cation of proteins from plants with unsequenced or incomplete genomes. As an example, 2D-DIGE was used in characterization of the strawberry proteome during ripening and developing stages, and correlation between different genotypes [ 39 ]. Another study employed 2D-DIGE to examine proteome expression changes of bark tissues of peach ( Prunus persica L. Batsch) in exposure to low temperature and short pho- toperiod [ 40 ]. Furthermore, the technique is still considered a useful tool to detect protein isoforms and modi ed proteins for which there are no effective and efcient enrichment strategies available [ 41 ]. To address some of the issues with 2DE, the higher throughput Mudpit tech- nique was introduced. Multi dimensional protein identi cation technique (MuDPIT) is a HPLC peptide separation method coupled with mass spectrometry which is more capable than 2-DE of identifying less abundant, basic, hydrophobic and membrane-spanning proteins [ 42 ]. Mudpit analysis requires that all proteins in a sample be digested into peptides before the separation steps. Differential compar- ative quantitation studies by Mudpit have been reported using isotope labeling in vitro [ 43 ] or in vivo [ 44 ]. One of the earliest reports in this area used Mudpit in parallel with 2-DE to characterize the proteome of rice leaf, root and seed tissues, which included the identi cation of more than 2500 unique proteins [ 45 ].

1.3.2 Stable Isotope Labelling (Chemical Metabolic)

Stable isotope labelling is a powerful quantitative proteomics approach, which has been used in a large number of plant studies. These techniques are divided into two major groups; chemical and metabolic labelling. In chemical labelling techniques (ICAT, iTRAQ, TMT and dimethyl labelling), differential isotopic labels are incorporated into the samples after protein extraction and preparation, whereas in

1

Applications of Quantitative Proteomics in Plant Research

7

metabolic labelling (SILAC and 15 N), labels are added to the growth media to be metabolised by the cell and ultimately label the whole organism.

1.3.3 Chemical Labeling

1.3.3.1 ICAT

One of the rst quantitative proteomics methods using chemical labeling reagents was isotope coded afnity tagging (ICAT) [ 46 ]. This is a thiol speci c proteomic technique in which protein samples are labeled with light or heavy versions of ICAT reagents on cysteine thiol groups. Samples are mixed and digested by an endoprotease such as trypsin. The relative abundance changes in the proteome can be obtained by comparing the intensities of labeled protein peaks with light and heavy mass tags. The technique is capable of comparing the protein expression changes between two biological samples. This approach has several drawbacks, including the fact that it is limited to proteins containing cysteine residues, and is less efcient at labeling acidic proteins. Applications of ICAT in plant proteomics have been reported only in a few studies, mainly focused on organelle membrane protein distribution such as characterizing the endoplasmic reticulum and Golgi apparatus in Arabidopsis [ 47 ], mitochondrial membrane proteins in Arabidopsis [ 48 ] and chloroplast soluble stromal proteins in maize leaves [ 49 ].

1.3.3.2 iTRAQ and TMT

Isobaric tags for relative and absolute quantitation (iTRAQ) [ 50 ] and tandem mass tag (TMT) [ 51 ] methods are designed based on isobaric labelling reagents. The multiplexing capabilities of these techniques provide an opportunity to compare the proteome of up to 8 samples in iTRAQ, and up to 10 samples in TMT, simulta- neously in a single MS analysis. The isobaric tags with the same mono-isotopic masses chemically label the N-terminus and the amine groups of the lysine side chain of tryptic peptides. Labelled peptides from different samples/conditions are combined, desalted, fractionated and analyzed using high mass accuracy mass spectrometers. Upon fragmentation of the tag attached to the peptides, reporter ion intensities are generated which are unique to the tags used in labelling of the peptides from different biological samples. One of the major advantages of multi- plexing is that MS1 complexity stays at a single proteome, whereas labelling strategies that use MS1 based quantitation (except label-free) become more com- plex in the MS1 as more labels are added. For example, SILAC or dimethylation labelling with light and heavy reagents are twice as complex. In multiplexing, all of the samples contribute to one peak. This contributes to depth of coverage. Hence, there is no further penalty for increased multiplexing capabilities.

8

M. Mirzaei et al.

The reporter ion intensities represent the relative abundance of the peptides and proteins from which they are originated. Both iTRAQ and TMT techniques bene t from their multiplexing power, however they have their own unique drawbacks such as the cost of the reagents, and the need for very high mass accuracy spec- trometers. Furthermore, specialised instrumentation is needed to handle the ability to purify co-isolated peptides using MS3 strategies [ 52 ]. The use of iTRAQ and TMT has been reported in a large number of plant quantitative studies in recent years (Fig. 1.1 ). For instance, iTRAQ was used to investigate the response mechanism of various plants to a wide range of stresses including cold [53 ], heat [ 54 ] drought [ 55 ] salinity [ 56 ], and heavy metals [ 57 ]. Similarly, TMT was used in comparative expression studies for a number of plant species such as Arabidopsis [ 58 , 59 ], rice [ 60 ], and barley [61 ]. The number of plant study reports using iTRAQ is signi cantly higher than TMT, mainly because iTRAQ was introduced a few years ahead of TMT. However, the higher multiplexing capability of TMT (TMT10plex compared to 8plex iTRAQ), has already attracted the attention of plant researchers and it is expected that the number of studies using TMT will rise over the next few years (Fig. 1.1 ).

1.3.4 Metabolic Labeling

The recent technological advances in proteomics enable the incorporation of stable isotope labels into samples to accurately detect changes in protein abundance [ 62 ]. Metabolic labelling refers to the methods in which stable isotopes are incorporated in vivo during the translation stage of the protein synthesis in cells [ 63 ]. The cells are grown in the media supplemented with the isotopes, so that they are incorpo- rated into the proteome metabolically. Mass spectrometric analysis is then per- formed on digested lysates, and quantitation is performed by distinguishing the mass shifts between light and heavy isotopes [ 64 , 65 ]. Metabolic labelling is in vivo labelling, and the different techniques of in vivo stable isotope labelling include 15 N labeling, Stable Isotope Labeling of Amino acids in Cell Culture (SILAC), 13 C labelling. In plant proteomics, 15 N and SILAC have been used to perform com- parative studies on different metabolic states of the plant cells [ 65 68 ].

1.3.4.1 SILAC

Stable Isotope Labelling by Amino acids in Cell culture (SILAC) is the in vivo incorporation of stable isotope-containing amino acids, such as arginine and lysine, that label proteins during their cellular synthesis [ 69 ]. The technique is highly efcient and reproducible and is considered a powerful tool for studying PTMs such as phosphorylation. SILAC has been successful in numerous yeast and mammalian studies [ 70 72 ]. However, this technique has not been as successful in plants.

1

Applications of Quantitative Proteomics in Plant Research

9

This might be due to poor labelling efciency of autotrophic plant cells which are able to synthesize all amino acids [65 , 73 ]. To date, there are only three SILAC based quantitation studies reported in plants. Two of these were carried on Arabidopsis cell culture [ 65 , 66 ] and the labelling efciency did not exceed more than 90 %. However, a recent study by Lewandowska et al. [ 74 ] demonstrated a technique to label the whole Arabidopsis seedlings using stable isotope-containing arginine and lysine with more than 95 % labelling efciency [ 74 ]. The reliability and efciency of this method remains to be con rmed in future studies. In general, drawbacks of any MS1 based quantitation strategy stem from higher false positive rates in the regulated set of proteins that are quanti ed from single peptide-based protein identi cations. This is because random assignments are more likely to have only one isotope identi ed. The chance of identifying both isotopes simultaneously for the same random peptide is much less likely.

1.3.4.2 15 N Labelling

Metabolic labelling with 15 N appears to be more suitable for plant studies due to the fact that nitrogen is usually a limiting factor in plant growth, and 15 N-containing salt as the sole nitrogen source can be added to plant cell cultures or the whole plant systems and be efciently incorporated into amino-acids and proteins. Metabolic labelling with the heavy isotope of nitrogen could be full or partial depending upon the amount of the heavy 15 N isotopes used. In full labelling, up to 98 % of the proteinaceous nitrogen is labelled with 15 N isotopes, while in partial labelling a lower percentages of the heavy nitrogen is incorporated.

1.3.4.3 Different Experimental Approaches of 15 N Labeling in Plants

Labeling with K 15 NO 3 has been carried out in various systems such as Arabidopsis cell culture [ 75 ] and whole Arabidopsis plants grown in liquid nutrient media using the hydroponic isotope labeling of entire plants (HILEP) [ 68 ]. In labeling the cells with 15 N, two populations of cells (control cells and treatment cells or normal cells and stress affected cells) are grown in two distinct media containing 15 N and 14 N. The two samples are mixed, processed and subjected to mass spectrometry analysis. The peptides from both the control and the treatment samples have the same chemical properties but exhibit mass difference because of the heavy isotope labeling that is detectable by the mass spectrometer [ 5 ]. On comparing the peak intensities between the two samples the difference in the expression of the proteins is analysed [ 1 ]. The different experimental strategies that have been used to analyse plants range from full to partial labelling techniques, reciprocal labeling and pulse labeling techniques. Several experiments were performed using 15 N-labeling in plants systems with the aim of developing the method on plants including, Glycine

10

M. Mirzaei et al.

max (Soyabean) [76 ], Arabidopsis thaliana [ 77 , 78 ], Solanum lycopersicum (Tomato) [ 79 ], Hordeum vulgare ) (Barley) [80 ].

1.3.4.4 Full 15 N-Labeling

Complete or full metabolic labeling refers to labeling all of the nitrogen in the cells with heavy nitrogen. The main challenge present in full metabolic labeling is achieving the efcient growth of an organism supplemented with N 15 entirely [ 78 ]. Successful full metabolic labeling for proteomic investigation was demonstrated with complete efciency of labeling intact plants in Arabidposis thaliana [ 81 ]. The plants to be labeled were grown in media containing 98 % of 15 NH 15 4 NO 3 and K 15 NO 3 in place of natural abundance salts. Complete incorporation of about 98 % of the heavy nitrogen was achieved in the Arabidopsis seedlings. The evaluation of the performance of MASCOT, a standard MS/MS search engine was also evaluated for the combined analysis of the 14 N and 15 N-labeled peptide samples of Arabidopsis seedlings which proves the application of 15 N-metabolic labeling for quantitative proteomics analysis with excellent incorporation [81 ].

1.3.4.5 Partial 15 N-Labeling

An alternative strategy to use isotope labeling was proposed by Whitelegge et al. [ 82 ] which was to decrease the amount of heavy nitrogen used for labeling resulting in partial labeling. It was reported that when both the natural and the partial labeled forms were combined, the shape of the resulting isotopic envelope is used to determine the relative amount of each peptide present in the sample mixture which enables information to be extracted from partial metabolic labeling rather than full metabolic labeling [ 82 ]. A comparison of full and partial metabolic labeling in Arabidopsis thaliana was made by Huttlin et al. [78 ]. In this experiment, labeled and unlabeled mixtures of Arabidposis peptides were rst analysed using both full and partial labeling, each technique was assessed for consistency, dynamic range and reproducibility under controlled conditions. Further analysis of light versus dark grown Arabidopsis using both the techniques was carried out to analyse the performance. It was found that with partial metabolic labeling allowed the more complete biological comparison of the protein expression that exhibited signi cant changes under conditions, where full metabolic labeling failed to give reliable quantitative information. Partial metabolic labeling also serves as a much more economic way of metabolic labeling [ 78 ] by decreasing the cost of the labelled nutrients, and allows the quanti cation of more peptides across the whole dynamic range. But, from the thorough comparison of the full and partial labeling tech- niques, partial metabolic labeling is more challenging in the automated identi ca- tion of labeled and unlabeled peptide pairs and in the quanti cation of the change in the isotope cluster distribution [ 83 ].

1

Applications of Quantitative Proteomics in Plant Research

11

1.3.5 Reciprocal Labeling

In a reciprocal labeling experimental strategy, two pools of samples for e.g. control and treatment samples or mutant and wild type samples, are inversely labeled with heavy nitrogen in such a way that the label is associated once with the treatment and once with sample and vice versa. Pair wise comparisons made from the mass spectrometric analysis would be used to evaluate the changes in the protein abundance [ 83 ]. In plant proteomics, reciprocal heavy metal labeling has been used to study stress physiology including the study of the effect on elicitors on protein phosphorylation [84 ], and microdomain composition of Arabidopsis and tobacco using cell cultures [ 85 , 86 ]. Whole-plant studies have been carried out to charac- terize the effect of abscisic acid treatment on phosphorylation [ 86 ], and protein abundance changes have been monitored during heat shock responses [ 68 ] and leaf senescence [87 ].

1.4 Label-Free Quantitation

Label free quantitation has become popular over the years mainly owing to its ease of use and application in a wide range of biological studies [26 , 88 ]. It is subdivided into two separate strategies; spectral counting (SC) [ 89 ] and area under the curve (AUC) [ 90 ]. In label free quantitative shotgun proteomics, both control and sample are subjected to separate LC-MS/MS analysis, and protein quantitation is performed on either peak intensity of the same peptide or the number of spectral counts for the same proteins [91 ]. In the SC approach the most abundant peptides will be selected for further fragmentation, hence the abundance of generated MS/MS spectra is proportional to the protein amount in data-dependent acquisition whereas in the AUC approach the protein abundances are estimated from the measurement of the changes in ion intensity of chromatographic peak areas or heights. All quantitative MS approaches have their own advantages and drawbacks. Some issues are common among all relative quantitative MS and MS/MS approaches, such as: accounting for peptides that are shared between different gene products or protein isoforms, and issues of missing peptide ions between biological replicates. Missing peptide ions are especially problematic for low abundance proteins that are close to the detection limit of the mass spectrometer. Label-free quantitation, whether based on the number of peptides for the spectral counts approaches, or peak intensity for the area under the curve (AUC) approaches, also has speci c limitations. The speci c drawbacks to spectral counting are that the actual relative fold changes in protein abundance are not easily calculated and the detection of differential protein abundance is difcult for proteins that generate low spectral counts (<5). In the case of AUC relative quantitation, there are certain concerns regarding variation in MS1 signal intensity and chemical and biological interferences between replicates due to changes in chromatography

12

M. Mirzaei et al.

performance from run-to-run. Despite these caveats, label free quantitative pro- teomics remains a versatile and practical approach for global proteome pro ling studies. For low mass accuracy instruments, including ion traps, spectral counting is the method of choice over AUC. However, label-free AUC quantitative work ows have been increasing in use, largely due to improved computational platforms and wider availability of high accuracy mass spectrometers. Label-free quantitative proteomics has been the method of choice over other techniques for the majority of the plant discovery experiments, mainly due to its affordability and ease of use features. In our lab we used label-free quantitation based on spectral counting in more than 20 different plant studies; for instance Gammulla et al. [92 , 93 ] investigated the changes in the proteome of rice leaves and cell cultures in response to high and low temperatures using a label-free based spectral counting approach in two separate studies. In both studies, SDS-PAGE was employed to fractionate proteins. Each lane was cut into 16 pieces, followed by in-gel digestion and MS analysis by nano ow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) using a linear ion-trap mass spectrometer. In another study, the effect of thermal stress on cabernet sauvignon grape cells was evaluated by a combination of Filter-aided sample preparation (FASP) digests and spectral counting [ 94 ]. The same technique was applied to understand the molecular mechanisms of water de cit stress in rice shoots [ 95 , 96 ] and roots [26 ], and mid-mature peanut seeds [ 97 ].

1.5 Post-translational Modi cations (PTMs) Plant Proteomics

PTMs are known to play a key role in controlling the activity and function of a wide range of proteins within a cell. Across both animal and plant kingdoms, we nd a grand diversity of post-translational modi cations (PTMs). There are about 400 discrete types of PTMs which can occur in a cell [ 98 ]. Each of the modi cations has the potential to signi cantly alter the conformational space of the protein and ultimately its function. Mass spectrometry is considered the most suitable tool for identifying novel post translational modi cations, as no prior information about the modi cation sites and type is essential [ 99 ]. Although the body of research into PTM function is growing, researchers have only just scratched the surface on uncovering the importance of these modi cations. The most commonly studied PTM is phosphorylation the ubiquitous on/off switch responsible for developmental pathways associated with cell growth and in response to stress factors. In addition, protein acetylation, methylation, ubiquiti- nation, glycosylation and a whole host of niche modi cations have been thoroughly studied and catalogued [ 100 103 ]. Recent large-scale plant quantitative proteomics

1

Applications of Quantitative Proteomics in Plant Research

13

experiments have successfully linked many PTMs to a wide range of metabolic functions and pathways operated during unfavourable conditions [ 104 ]. It is therefore important to have knowledge of what these modi cations are, how they affect protein structure and function, and how they can contribute to some important downstream biological phenotypes. The aim of this chapter is not to list in detail every example of where a certain type of modi cation exists, but to provide an overview of the most common types of modi cations occurring in plant proteins, and highlight their importance in plant development and survival.

1.5.1 Phosphorylation

Phosphorylation remains the most widely recognised and studied PTM. Simply put, phosphorylation is the addition of a phosphate group (PO 2 ), usually on the hydroxyl group of threonine, tyrosine, histidine or serine residues, which increases the molecular weight by 79.9663 Da [ 105 ]. An overwhelming number of large-scale phospho-proteomics studies have been reported in rice, Arabidopsis , Medicago, maize, and several other plant species, which were carefully discussed in recent reviews [ 106 108 ]. Phosphorylation can be thought of as an on/off signal mechanism; proteins that are phosphorylated have phosphate groups added to speci c amino acids sites to become activated . In reality, adding a phosphate group may have many different consequences, and isn t simply con ned to turning proteins on or off . For example, the addition of a phosphate group to a protein may provide it with a new tertiary structure, conferring a new active site and thus a new function. In providing new secondary or tertiary conformations for the protein, other important functions such as protein-protein interactions may be promoted, or be interfered with or shut down [ 109 ]. It is important to remember that phosphorylation also includes the counter-process: the removal of phosphate groups. Phosphatases (removal) play just as vital a role in protein function as protein kinases (addition), in situations where the phosphate group regulates signalling mechanisms between proteins. Larger scale biological processes are also dependant on kinase activity and phosphoryla- tion and these include differentiation, cell maintenance, development and intra- cellular regulation [ 110 ]. For the purpose of this overview, it is important to consider the impact of phosphorylation in the plant research eld. As we have established above, changes to the protein at the phosphate level can impact cell growth and development due to the switching on of various chemical pathways. In addition, phosphorylation also plays a critical role in host-pathogen responses and can inuence plant survival [ 111 ]. Some examples of current phosphorylation-related research include the role of phosphorylation in controlling mitochondrial related processes, especially related to an enzyme responsible for acetyl-CoA processing within the TCA cycle [ 112 ];

3

14

M. Mirzaei et al.

for cell wall elasticity and cell cycle regulation within Phaseolus vulgaris (common bean) [ 113 ]; and for elucidating the relationship between classes of proteins such as aquaporins in the water-moving capabilities of roots [ 114 ].

1.5.2 Acetylation

The post-translational acetylation of lysine residues on speci c proteins is a func- tional regulatory mechanism, which was only discovered relatively recently [ 115 ]. It is a topic of great research interest, because it seems to be universal in nature; it is present across all the kingdoms of life [ 116 , 117 ]. Plant proteins are known to be subjected to acetylation and deacetylation by acetyltransferases and deacetylases, which have been identi ed in the genome of various plant species. At least twelve histone acetyltransferases (HAT) and eighteen histone deacetylases (HDAC) are present in the Arabidopsis genome [ 118 ], while the rice genome contains 19 HDAC genes and seven HAT genes [ 119 ]. Histone acetylation plays a signi cant role in the regulation of cell cycle, development, owering time, and hormone signal transduction [ 120 ]. Despite the extensive studies on dynamic and reversible changes in histone acetylation in histones, the extent of lysine acetylation in non-histone proteins in plant cells is largely unknown. A number of published studies in different species across the animal kingdoms have shown that acetylation is an important regulatory mechanism [ 121 ]. Some of the earliest relatively large scale studies of lysine acetylation were performed in Arabidopsis : Konig et al. [122 ] reported the identication of 120 lysine acetylated mitochondrial proteins, containing 243 distinct sites of acetylation, mainly on proteins involved in protein metabolism and the tricarboxylic acid cycle; Finkemeier et al. [ 100 ] published the identication of 91 acetylated sites on 74 proteins representing diverse functional classes; and Wu et al. [ 123 ] identi ed 64 lysine acetylation sites on 57 proteins and showed that lysine acetylation is an important factor in the regulation of key metabolic enzymes. In the latter study, the authors identi ed acetylated proteins including photosystem II (PSII) subunits, light-harvesting chlorophyll a/b-binding proteins, RuBisCO large and small sub- units, and chloroplastic ATP synthase ( b-subunit) [ 123 ]. Similar studies have subsequently been reported in pea [ 124 ], soya bean [ 125 ], grapevine [ 126 ], and rice [ 127 ]. All of these studies have shown that acetylated proteins in plant cells are not limited to histones and seem to be involved in a diverse range of metabolic control and energy-related functions, and found in a diverse range of cellular compartments. However, the studies published so far have analysed plant cells under ideal growing conditions; none of them have addressed the question of how the acetylation status of the peptides and proteins changes in response to the imposition of external stresses. Complex regulatory networks control protein expression in all cells. The chloroplast and mitochondria are known to be the main cellular hub for the con- version of energy and redox homeostasis in plant cells. Hence, control of protein

1

Applications of Quantitative Proteomics in Plant Research

15

functions in these important organelles is increasingly being found to be under- pinned by reversible post-translational modi cations like acetylation. It is known that environmental stresses, as well as changes in cellular nutrient availability or energy status, have a direct impact on global changes in mitochondrial protein acetylation [128 ]. It has been reported that over one third of proteins in mito- chondria are acetylated, of which the majority are associated with pathways such as signal transduction, histone/chromatin gene expression, or protein turnover [ 129 ]. Therefore, investigation of protein acetylation has emerged as an important research topic.

1.5.3 Methylation

Most scientists would be familiar with the notion of DNA methylation that is, the silencing of speci c genes by the addition of methyl groups on CpG sites, thus preventing transcription by structural interference with the major groove of DNA. With the discovery of the arginine methyltransferase family of proteins, protein methylation is now considered another form of PTM that warrants examination. Methylation of key arginines within proteins, such as ribosome binding proteins, has been shown to interfere with the intermolecular forces present within the binding site, thus reducing protein-protein afnity [ 130 ]. Other key areas where methylation of arginines has been shown to impact on protein function include proteins responsible for transcriptional regulation, signal transduction and even DNA repair [ 130 , 131 ]. However, it is also important to note that methylation is not limited to arginine residues and has been found on many other amino acid sites; but again, the overall effect of these modi cations change the electrostatic potential of speci c sites within the protein structure, which has downstream consequences for pathway function. Recent examples of methylation research include the localization of a methy- lation enzyme responsible for regulation of a ribosomal protein to chloroplasts and mitochondria [101 ]. There is also a recent push to characterize the processes by which N-terminal modi cations occur, as there is mounting evidence that even rare N-terminals that end with Arg residues can be subject to protein methylation [ 132 ]. In fact, N-terminal modi cation is an area of research that could warrant an entire review, given the functional importance and diversity of associated PTMs.

1.5.4 Ubiquitination

Ubiquitination is the process by which proteins are tagged for degradation. Protein ubiquitination involves of the covalent and reversible binding, of a single ubiquitin molecule or a number of ubiquitin molecules to Lys residues of the target protein [ 133 ]. Ubiquitination is carried out by addition of ubiquitin protein by a set of three

16

M. Mirzaei et al.

enzymes the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme E2, and ubiquitin protein ligaseE3. Ubiquitination is a well-observed process that spans both animal and plant kingdom. Effectively, ubiquitin transferases mark misfolded or excess proteins with an ubiquitin tag; ubiquinated structures are then read by the proteosome (a complex multimer that acts a bit like a molecular grinder) and the tagged protein is degraded into oligopeptide chains. New research, however, suggests that ubiquitination is not solely for protein degradation and actually has a role in modulating biochemical pathways [ 134 ]. In terms of the importance of ubiquitination, research groups have linked this particular PTM with pathogen defense and development structures [ 135 , 136 ]. This underscores an important point that the major function of a PTM is not the sole function, and ubiquitin is no exception, as highlighted by research showing the implications of ubiquitin patterning for the life cycle and regulation of plant cells [ 137 ]. As such, ubiquitination has been found to control subcellular processes such as localization and cross talk [ 138 ]. Hence, this important PTM has a crucial role in plant metabolism, growth and development, hormone signalling and stress response to wide range of abiotic and biotic factors [139 , 140 ]. Recent research on Arabidopsis thaliana has uncovered over three thousand individual ubiquitination sites over sixteen hundred proteins [ 102 ]. Another pro- teomics study showed that 950 ubiquitination substrates in whole Arabidopsis thaliana seedlings were identi ed, using stringent two-step afnity methods for purifying Ub-protein conjugates [ 141 ]. Ubiquitination is also being investigated for its role in the regulation of complex biomolecular pathways, as a push from the latter half of last decade saw a newfound interest in investigating alternative ubiquitin roles [138 , 142 , 143 ].

1.5.5 Glycosylation

Protein glycosylation, however, is a different PTM class altogether. This is an enormous eld of study that is encompassed by the term glycomics, which is complementary to metabolomics, proteomics and genomics. At its core, glycomics refers to the study of sugar modi cation that exist on proteins, namely O- and N-linked glycans, so named because of the different type of bond and hence amino acid to which the glycan is attached. In order to study glycans or glycopeptide structures, one must invest a considerable amount of time and effort as there exists no easy automated approach to understanding the structures of sugar additions based on mass spectrometry pro les. This is due to the nature of sugar branching from amino acids even if the constituent sugar modules can be singularly iden- ti ed, there is as yet no method of automatic determination to reassemble these monomers into accurate morphologies [ 144 ]. Sugar modi cations often change the function of proteins in subtle ways; for instance, surface membrane proteins coated in sugars act as intracellular commu- nication devices that can recognise self versus antigen molecules. It may be

1

Applications of Quantitative Proteomics in Plant Research

17

pertinent to mention that for intracellular protein modi cations that use O-linked glycans, the overall effect on protein function may be similar to that of phospho- rylation [ 103 , 145 ]. However, studying glycan patterns requires speci c expertise, in no small part due to the inherent difculties in acquiring glycan samples from peptides, and the manual annotation analysis that needs to take place to make sense of any glycosylation results. A recent overview of cell wall glycoproteins outlines the sparse discoveries for glycomics in Arabidopsis thaliana ; current knowledge is limited to an O-linked class of proteins in the cell wall, and a few N-linked glycans responsible for regulation of peroxidases, a mannosidase and a polygalacturonase inhibiting protein [ 146 ]. It is clear that glycomics lags behind other PTMs in plant research, hence more research needs to done to reveal the importance of glycomics in plant development and stress response studies.

1.5.6 PTMs Cross-Talk

The reversible nature of most PTMs makes some speci c residues subject to modi cation with alternate PTMs. In fact, a rst PTM can initiate the occurrence of the next PTMs. As a result the cross talk between PTMs occur on the same protein. Broadly speaking, PTM cross talk can be broken down into two easy-to-understand categories. First, positive cross talking occurs when the initial PTM make a con- structive modi cation, such as the addition of another nearby PTM, or if the initial PTM itself forms a new binding partner with another protein. Second, negative cross talk can also occur; in this instance, the presence of a single PTM can overcrowd the target site, change the conformation of the protein to stop other PTMs from forming, or even disable or inconvenience the functioning of a pre-established PTM [ 147 ]. Cross talk analysis is effectively the coalface of func- tional analysis, as often the functional result of PTMs on these proteins or signals come down to the interplay between different PTM types, which is in uenced by their location and numbers. Although preparation of tissue samples for PTM analysis can be tedious and costly, especially in the case of the phosphorylation or glycan studies, any solid understanding of intracellular regulation in response to the environment or stress factors must take PTMs into account. Having gone to the effort of preparing protein samples for PTM analysis it also makes sense to conduct PTM studies in addition to shotgun or bottom-up proteomics experiment to quantify the global protein changes within the cell. Having these two pieces of information hand in hand provides a rigorous and holistic picture of the cell s response to the factor in question the large-scale changes of protein expression, or presence and absence of proteins, informs us of the fundamental changes, while PTMs provide insight on how the cell responds in a more subtle and nuanced sense. Sometimes, these two pieces of information can overlap in addition to fold changes, we see PTM to the proteins of interest but often the changes on the PTM level may come from proteins that

18

M. Mirzaei et al.

are otherwise stable expression-wise but that alternate between two states. In this sense, an alteration in expression level cannot tell us anything about PTMs and vice versa.

1.6 Bioinformatics, Data Analysis Challenges and Platforms in the Plant Proteomics Field

The quantitative protein identication strategies described above rely on matching spectra to sequences from a database in order to generate the protein identication and quantitation; as such it is crucial to have a good quality database. If the plant in question is sequenced and well-characterized the choice is easy. For non-sequenced plants, the options range from using a limited species-specic database, a related sequenced and well annotated species, available EST databases, a comprehensive database such as NCBI plants, or smaller composite databases containing sequences from several related plants; the choices are well described in the context of plants in a recent review [148 ]. The majority of the papers surveyed there used the com- prehensive NCBI plant database; from the remaining options, from amongst the several papers carrying out comparative experiments, no clear winner option has emerged, with some nding the well-annotated related species more advantageous, others the species-speci c [ 149 ] or EST option [ 150 ]. From work in our own group on bioinformatics approaches available for wheat proteomic analyses [ 151 ], we found a smaller size database from a closely related species to be more manageable for multi-run iTRAQ experiments. We likewise emphasized the importance of considering issues such as database redundancy and the implication on protein grouping, as well as the availability of down-stream analysis options when choosing the database. Viewed from the plant biology standpoint the quantitative proteomics approa- ches are not a goal in themselves but a means to arrive at a useful biological or agricultural outcome. Therefore, a quantitative dataset generated by any of the proteomic technologies described above is just the beginning. The aim is increas- ingly not only placing the proteomic results in the context of biological information, but also gaining understanding of the mechanism of a particular process, be it drought response, signalling, or pathogen resistance. Here, we give an overview of commonly used tools and strategies for bioinformatics analysis of plant proteomics datasets. For selective reviews with a focus on bioinformatics resources available for plants across several Omics, platforms, see [ 152 ] and [ 153 ]. The broad outline of possible analysis steps is summarized in (Fig. 1.2 ), where for clarity we structure the work ow in three layers: data, tools and annotation, and results and interpretation. The particular details depend crucially of the type of plant, availability of annotations, and thus resources at ones disposal. Following experiment-speci c statistical analysis, all the quantitative results have to be placed in the context of available biological knowledge, such as pathways or biological

1

Applications of Quantitative Proteomics in Plant Research

19

of Quantitative Proteomics in Plant Research 19 Fig. 1.2 Schematic representation illustrating the plant

Fig. 1.2 Schematic representation illustrating the plant proteomics data analysis work ow

functions. If the organism is a well-characterised plant such as Arabidopsis or rice, then no further mapping of the protein identi ers is needed. If an organism has little or no annotation available then mapping the identi ers to commonly used orthologs such as Uniprot ( http://www.uniprot.org/mapping/ ), or BLAST mapping to a well-annotated species, may be a useful rst step. In the tools layer, the most convenient option is to use an analysis portal that integrates functional or pathway data with some options for analysis, provided that the plant species used is amongst those supported. KEGG remains a popular portal [ 154 ] containing pathway information for approximately 50 plant species, with the full list of supported organisms available at http://www.genome.jp/kegg/catalog/ org_list4.html . The KEGG Mapper gives the option to bulk map identi ers onto pathway images, for a short list of available types of protein ID s (KEGG, NCBI and Uniprot); other identi ers could be rst mapped to Uniprot as described above. Alternatively, bioinformatics portals such as KOBAS [ 155 ] ( http://kobas.cbi.pku. edu.cn/home.do ) integrate the KEGG database with other resources such as addi- tional databases like PANTHER, and gene ontology (GO) information, from a total of 1327 species. The AgriGO analysis platform [ 156 ] ( http://bioinfo.cau.edu.cn/ agriGO/analysis.php ) is a plant-speci c resource that integrates gene ontology data available at the moment for 45 plant species with six analyses options available, of which parametric gene set analysis (PAGE) can be used to integrate the abundance data from an experiment alongside the gene ontology information. The STRING [ 157 ] database and analysis portal has increased in popularity; while primarily used

20

M. Mirzaei et al.

for visualisation of protein interactions, it also contains gene ontology and pathway information that can be overlayed on the networks. If the plant of interest is not amongst those directly supported by one of the available analysis portals, or if a different type of analysis is required, then either software or annotation can be installed or downloaded separately. Tools such as Mapman for the analysis of pathway information [ 158 ], or Cytoscape ( http://www. cytoscape.org/) for the analysis of protein networks, have matured, and their installation and use is now much more user-friendly. Mapman can integrate and visualise experimental data, for instance across a time course, but coming from the proteomics perspective it still requires downloading the right mappings from the MapMan Store, and possibly blasting the protein identiers to the available map- ping identi ers; this can be done for instance via the Plant Expression Database Blast resource (http://www.plexdb.org/ ). Cytoscape plugins such as BinGO [ 159 ] and ClueGO [ 160 ] offer the option to view the gene ontology data in network form. For organisms with very limited information available an option might be a gene ontology information download, which can be explored using tools such as the WEGO portal ( http://wego.genomics.org.cn/cgi-bin/wego/index.pl) [ 161 ]. From our own group, we use PloGO [162 ] for integrating GO data with protein abun- dance data in the context of complex experiments. For commonly used protein identi ers such as Ensembl or Uniprot, or when mappings to these identi ers can be generated as previously described, tools such as the Ensembl Biomart or the Uniprot retrieve/mapping service can be readily used to bulk-download gene ontology information. When other identi ers are used, organism-speci c resources may provide other options. The TAIR Arabidopsis portal maintains a list of plant model organism databases and resources, available at https://www.arabidopsis.org/ portals/genAnnotation/other_genomes/index.jsp . The established tools described above are geared towards the analysis of lists of proteins arising from proteomics experiments, but they are not directly applicable to the analysis of post translational modi cations. PTM analysis in plants, whilst growing in popularity, is not yet mature [ 163 ], with data repositories emerging for a limited number of well-studied plants, and limited options for analysis. Large studies lead to identications of PTM sites on peptides, which can be mapped onto protein sequences that can then be categorized as above in terms of gene ontology or pathway. One of the main roles of PTM experiments at this point is to accu- mulate information to be fed back into data repositories [ 164 ]. In the realm of plant studies, phosphorylation resources were established initially for several model plants (PhosPhAt, Medicago PhosphoProtein database), and more recently P3DB [ 165 ], currently containing phospho-site information for eight plants, but pre- dominantly Arabidopsis , Medicago and rice. While the variety of available software and resources is immense, the output of the tools and annotation layer is usually a dataset with appended biological infor- mation from various biological categories of interest. Making use of this infor- mation can range from descriptive to mechanistic. At the descriptive end, summaries of categories such as biological process or pathway and their visuali- sation can be provided for a set of proteins, or compared between various sets.

1

Applications of Quantitative Proteomics in Plant Research

21

Enrichment analysis is described in detail in a recent review [ 166 ], and is embedded into many of the tools and portals described above; broadly it compares annotation from a protein set of interest to a baseline and suggests interesting processes or pathways to further focus on. PTM analyses in plants are in part still descriptive at this point, as information is being accumulated and stored, but can be coupled with enrichment analysis to nd processes that are, for example, enriched in phospho- rylation sites, or to interrogate the conservation of PTM sites in orthologs [ 108 ], across plant species. Also, targeted questions can be asked by focusing on PTM sites on protein families of interest such as families conferring resistance against pathogens [ 108 ]. Finally, analyses of protein networks and interactions are geared towards exploring relationships between proteins with the aim of delivering knowledge and a more mechanistic understanding.

1.7 Conclusions and Future Directions

Plant proteomics methodology has progressed rapidly, with numerous studies now available characterising plant proteomes in great detail. A key success of such studies has been the use of quantitative protein abundance information to shape understanding of plant biological processes, particularly environmental stress responses. With these successes and the advent of new high throughput and more sensitive technologies, we expect to see an explosion in the near future in the use of quantitative proteomic techniques addressing plant biology questions (Fig. 1.1 ). Also apparent from this review of the literature is the fact that PTM analysis of plant proteomes is still in its infancy. This is particularly true for glycoproteome analysis which is at present a poorly understood and characterised PTM in plants, especially compared with PTM analysis of proteomes from other branches of the tree of life. Better methods to understand and characterise PTMs in plants are needed, and the development of these methods should be a priority. Finally, bioinformatic methods and databases speci c to plants need to be better described and utilised. It is clear that many databases and tools are adapted from mammalian and bacterial analysis systems, and these may not be ideal for analysis of plant proteomic data. In addition, there seems to be a gap in the literature between what bioinformatics predicts from the data analysis of proteomic data, and experimental veri cation of these predictions.

References

1. Cho WCS (2007) Proteomics technologies and challenges. Genomics Proteomics Bioinform 5:77 85 2. Patterson SD, Aebersold RH (2003) Proteomics: the rst decade and beyond. Nat Genet 33:311 323

22

M. Mirzaei et al.

3.

Jorr ín-Novo JV, Pascual J, S ánchez-Lucas R, Romero-Rodr íguez MC, Rodr íguez-Ortega MJ, Lenz C et al (2015) Fourteen years of plant proteomics re ected in proteomics: moving from model species and 2DE-based approaches to orphan species and gel-free platforms. Proteomics 15:1089 1112

4.

Pfannschmidt T, Ogrzewalla K, Baginsky S, Sickmann A, Meyer HE, Link G (2000) The multisubunit chloroplast RNA polymerase A from mustard (Sinapis alba L.). Eur J Biochem 267:253 261

5.

Chen S, Harmon AC (2006) Advances in plant proteomics. Proteomics 6:5504 5516

6.

Vanderschuren H, Lentz E, Zainuddin I, Gruissem W (2013) Proteomics of model and crop plant species: Status, current limitations and strategic advances for crop improvement. J Proteomics 93:519

7.

Carpentier SC, Panis B, Vertommen A, Swennen R, Sergeant K, Renaut J et al (2008) Proteome analysis of non-model plants: a challenging but powerful approach. Mass Spectrom Rev 27:354 377

8.

Kaul S, Koo HL, Jenkins J, Rizzo M, Rooney T, Tallon LJ et al (2000) Analysis of the genome sequence of the owering plant Arabidopsis thaliana . Nature 408:796 815

9.

Goff SA, Ricke D, Lan T-H, Presting G, Wang R, Dunn M et al (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296:92 100

10.

Armengaud J, Trapp J, Pible O, Geffard O, Chaumot A, Hartmann EM (2014) Non-model organisms, a species endangered by proteogenomics. J Proteomics 105:518

11.

Canovas FM, Dumas-Gaudot E, Recorbet G, Jorrin J, Mock HP, Rossignol M (2004) Plant proteome analysis. Proteomics 4:285298

12.

Jorr ín JV, Maldonado AM, Castillejo MA (2007) Plant proteome analysis: a 2006 update. Proteomics 7:2947 2962

13.

Abril N, Gion J-M, Kerner R, Mü ller-Starck G, Cerrillo RMN, Plomion C et al (2011) Proteomics research on forest trees, the most recalcitrant and orphan plant species. Phytochemistry 72:1219 1242

14.

Bindschedler LV, Cramer R (2011) Quantitative plant proteomics. Proteomics 11:756 775

15.

Junqueira M, Spirin V, Balbuena TS, Thomas H, Adzhubei I, Sunyaev S et al (2008) Protein identi cation pipeline for the homology-driven proteomics. J Proteomics 71:346 356

16.

Waridel P, Frank A, Thomas H, Surendranath V, Sunyaev S, Pevzner P et al (2007) Sequence similarity-driven proteomics in organisms with unknown genomes by LC-MS/MS and automated de novo sequencing. Proteomics 7:2318 2329

17.

Primmer CR, Papakostas S, Leder EH, Davis MJ, Ragan MA (2013) Annotated genes and nonannotated genomes: cross-species use of gene ontology in ecology and evolution research. Mol Ecol 22:3216 3241

18.

Jorrin-Novo JV (2014) Plant proteomics methods and protocols. Springer, New York

19.

Ma B, Johnson R (2012) De novo sequencing and homology searching. Mol Cell Proteomics

11(O111):014902

20.

Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. Hum Genet 26:231 243

21.

Ofarrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:40074021

22.

Bae MS, Cho EJ, Choi EY, Park OK (2003) Analysis of the Arabidopsis nuclear proteome and its response to cold stress. Plant J 36:652 663

23.

Jiang Y, Yang B, Harris NS, Deyholos MK (2007) Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots. J Exp Bot 58:35913607

24.

Aghaei K, Ehsanpour AA, Shah AH, Komatsu S (2009) Proteome analysis of soybean hypocotyl and root under salt stress. Amino Acids 36:9198

25.

Qiu QS, Huber JL, Booker FL, Jain V, Leakey AD, Fiscus EL et al (2008) Increased protein carbonylation in leaves of Arabidopsis and soybean in response to elevated [CO 2 ]. Photosynth Res 97:155 166

1

Applications of Quantitative Proteomics in Plant Research

23

26. Mirzaei M, Soltani N, Sarhadi E, Pascovici D, Keighley T, Salekdeh GH et al (2011) Shotgun proteomic analysis of long-distance drought signaling in rice roots. J Proteome Res 11:348 358

27. Sarhadi E, Bazargani MM, Sajise AG, Abdolahi S, Vispo NA, Arceta M et al (2012) Proteomic analysis of rice anthers under salt stress. Plant Physiol Biochem 58:280 287

28. Han C, Yang P, Sakata K, Komatsu S (2014) Quantitative proteomics reveals the role of protein phosphorylation in rice embryos during early stages of germination. J Proteome Res 13:1766 1782

29. Majoul T, Bancel E, Triboi E, Ben Hamida J, Branlard G (2004) Proteomic analysis of the

effect of heat stress on hexaploid wheat grain: characterization of heat-responsive proteins from non-prolamins fraction. Proteomics 4:505513

30. Faghani E, Gharechahi J, Komatsu S, Mirzaei M, Khavarinejad RA, NajaF et al (2015) Comparative physiology and proteomic analysis of two wheat genotypes contrasting in drought tolerance. J Proteomics 114:115

31. An Nguyen TT, Michaud D, Cloutier C (2007) Proteomic pro ling of aphid Macrosiphum euphorbiae responses to host-plant-mediated stress induced by defoliation and water decit.

J Insect Physiol 53:601 611

32. Aghaei K, Ehsanpour AA, Komatsu S (2008) Proteome analysis of potato under salt stress.

J Proteome Res 7:4858 4868

33. Yumiko I, Hiroshi H (2000) Effect of heat stress on tomato fruit protein expression. Electrophoresis 21:1766 1771

34. Bandehagh A, Salekdeh GH, Toorchi M, Mohammadi A, Komatsu S (2011) Comparative proteomic analysis of canola leaves under salinity stress. Proteomics 11:19651975

35. Taheri F, Nematzadeh G, Zamharir MG, Nekouei MK, Naghavi M, Mardi M et al (2011) Proteomic analysis of the Mexican lime tree response to Candidatus Phytoplasma aurantifolia infection. Mol BioSyst 7:3028 3035

36. Douce R, Joyard J (1990) Biochemistry and function of the plastid envelope. Annu Rev Cell Biol 6:173216

37. Yan JX, Harry RA, Spibey C, Dunn MJ (2000) Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes. Electrophoresis 21:3657 3665

38. Rabilloud T, Lelong C (2011) Two-dimensional gel electrophoresis in proteomics: a tutorial.

J Proteomics 74:18291841

39. Bianco L, Lopez L, Scalone AG, Di Carli M, Desiderio A, Benvenuto E et al (2009) Strawberry proteome characterization and its regulation during fruit ripening and in different genotypes. J Proteomics 72:586 607

40. Renaut J, Hausman JF, Bassett C, Artlip T, Cauchie HM, Witters E et al (2008) Quantitative proteomic analysis of short photoperiod and low-temperature responses in bark tissues of peach ( Prunus persica L. Batsch). Tree Genet Genomes 4:589600

41. Rogowska-Wrzesinska A, Le Bihan M-C, Thaysen-Andersen M, Roepstorff P (2013) 2D gels still have a niche in proteomics. J Proteomics 88:4 13

42. Washburn MP, Wolters D, Yates JR III (2001) Large-scale analysis of the yeast proteome by multidimensional protein identi cation technology. Nat Biotechnol 19:242 247

43. Chen R, Pan S, Yi EC, Donohoe S, Bronner MP, Potter JD et al (2006) Quantitative proteomic pro ling of pancreatic cancer juice. Proteomics 6:3871 3879

44. De Godoy LM, Olsen JV, De Souza GA, Li G, Mortensen P, Mann M (2006) Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system. Genome Biol 7:R50

45. Koller A, Washburn MP, Lange BM, Andon NL, Deciu C, Haynes PA et al (2002) Proteomic survey of metabolic pathways in rice. Proc Natl Acad Sci USA 99:11969 11974

46. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R (1999) Quantitative analysis of complex protein mixtures using isotope-coded afnity tags. Nat Biotechnol 17:994 999

24

M. Mirzaei et al.

48. Hartman NT, Sicilia F, Lilley KS, Dupree P (2007) Proteomic complex detection using sedimentation. Anal Chem 79:2078 2083

49. Majeran W, Cai Y, Sun Q, Van Wijk KJ (2005) Functional differentiation of bundle sheath and mesophyll maize chloroplasts determined by comparative proteomics. Plant Cell 17:3111 3140

50. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S et al (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154 1169

51. Thompson A, Sch äfer J, Kuhn K, Kienle S, Schwarz J, Schmidt G et al (2003) Tandem mass

tags: a novel quanti cation strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895 1904

52. Erickson BK, Jedrychowski MP, Mcalister GC, Everley RA, Kunz R, Gygi SP (2015) Evaluating multiplexed quantitative phosphopeptide analysis on a hybrid quadrupole mass lter/linear ion trap/orbitrap mass spectrometer. Anal Chem 87:1241 1249

53. Neilson KA, Mariani M, Haynes PA (2011) Quantitative proteomic analysis of cold-responsive proteins in rice. Proteomics 11:1696 1706

54. Liu G-T, Ma L, Duan W, Wang B-C, Li J-H, Xu H-G et al (2014) Differential proteomic analysis of grapevine leaves by iTRAQ reveals responses to heat stress and subsequent recovery. BMC Plant Biol 14:1

55. Hu X, Li N, Wu L, Li C, Li C, Zhang L et al (2015) Quantitative iTRAQ-based proteomic analysis of phosphoproteins and ABA-regulated phosphoproteins in maize leaves under osmotic stress. Sci Rep 5

56. Xu J, Lan H, Fang H, Huang X, Zhang H, Huang J (2015) Quantitative proteomic analysis of the rice (Oryza sativa L.) salt response. PLoS ONE 10:e0120978

57. Fukao Y, Ferjani A, Tomioka R, Nagasaki N, Kurata R, Nishimori Y et al (2011) iTRAQ analysis reveals mechanisms of growth defects due to excess zinc in Arabidopsis . Plant Physiol 155:18931907

58. Parker J, Zhu N, Zhu M, Chen S (2012) Proling thiol redox proteome using isotope tagging mass spectrometry. JoVE (J Visualized Exp) 61:e3766 e3766

59. Turek I, Wheeler JI, Gehring C, Irving HR, Marondedze C (2015) Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides. Data Brief 4:336 343

60. Neilson KA, Scafaro AP, Chick JM, George IS, Van Sluyter SC, Gygi SP et al (2013) The inuence of signals from chilled roots on the proteome of shoot tissues in rice seedlings. Proteomics 13:1922 1933

61. Zeng J, He X, Quan X, Cai S, Han Y, Nadira UA et al (2015) Identi cation of the proteins associated with low potassium tolerance in cultivated and Tibetan wild barley. J Proteomics 126:111

62. Gouw JW, Tops BB, Mortensen P, Heck AJ, Krijgsveld J (2008) Optimizing identi cation and quantitation of 15N-labeled proteins in comparative proteomics. Anal Chem 80:7796

7803

63. Wang Y, Li H, Chen S (2010) Advances in quantitative proteomics. Front Biol 5:195203

64. Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422:198207

65. Gruhler A, Schulze WX, Matthiesen R, Mann M, Jensen ON (2005) Stable isotope labeling of Arabidopsis thaliana cells and quantitative proteomics by mass spectrometry. Mol Cell Proteomics 4:1697 1709

66. Sch ütz W, Hausmann N, Krug K, Hampp R, Macek B (2011) Extending SILAC to proteomics of plant cell lines. Plant Cell 23:17011705

67. Engelsberger WR, Erban A, Kopka J, Schulze WX (2006) Metabolic labeling of plant cell cultures with K 15 NO 3 as a tool for quantitative analysis of proteins and metabolites. Plant Methods 2:1

1

Applications of Quantitative Proteomics in Plant Research

25

69. Oda Y, Huang K, Cross FR, Cowburn D, Chait BT (1999) Accurate quantitation of protein expression and site-speci c phosphorylation. Proc Natl Acad Sci 96:65916596

70. Ong S-E, Mann M (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc 1:2650 2660

71. Hulce JJ, Cognetta AB, Niphakis MJ, Tully SE, Cravatt BF (2013) Proteome-wide mapping of cholesterol-interacting proteins in mammalian cells. Nat Methods 10:259 264

72. Nagaraj N, Kulak NA, Cox J, Neuhauser N, Mayr K, Hoerning O et al (2012) System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top Orbitrap. Mol Cell Proteomics 11(M111):013722

73. Hirner A, Ladwig F, Stransky H, Okumoto S, Keinath M, Harms A et al (2006) Arabidopsis LHT1 is a high-afnity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll. Plant Cell 18:1931 1946

74. Lewandowska D, Ten Have S, Hodge K, Tillemans V, Lamond AI, Brown JWS (2013) Plant SILAC: stable-isotope labelling with amino acids of Arabidopsis seedlings for quantitative proteomics. PloS ONE 8:e72207

75. Engelsberger WR, Erban A, Kopka J, Schulze WX (2006) Metabolic labeling of plant cell cultures with K 15 NO 3 as a tool for quantitative analysis of proteins and metabolites. Plant Methods 2:14

76. Allen DK, Evans BS, Libourel IGL (2014) Analysis of isotopic labeling in peptide fragments by tandem mass spectrometry. PLoS ONE 9:e91537

77. Lanquar V, Kuhn L, Leliè vre F, Khaf M, Espagne C, Bruley C et al (2007) 15N-Metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells. Proteomics 7:750754

78. Huttlin EL, Hegeman AD, Harms AC, Sussman MR (2007) Comparison of full versus partial metabolic labeling for quantitative proteomics analysis in Arabidopsis thaliana . Mol Cell Proteomics 6:860 881

79. Schaff JE, Mbeunkui F, Blackburn K, Bird DM, Goshe MB (2008) SILIP: a novel stable isotope labeling method for in planta quantitative proteomic analysis. Plant J 56:840 854

80. Nelson CJ, Alexova R, Jacoby RP, Millar AH (2014) Proteins with high turnover rate in barley leaves estimated by proteome analysis combined with in planta isotope labeling. Plant Physiol 166:91 108

81. Nelson CJ, Huttlin EL, Hegeman AD, Harms AC, Sussman MR (2007) Implications of 15N-metabolic labeling for automated peptide identi cation in Arabidopsis thaliana. Proteomics 7:1279 1292

82. Whitelegge JP, Katz JE, Pihakari KA, Hale R, Aguilera R, G ómez SM et al (2004) Subtle modi cation of isotope ratio proteomics; an integrated strategy for expression proteomics. Phytochemistry 65:1507 1515

83. Arsova B, Kierszniowska S, Schulze WX (2012) The use of heavy nitrogen in quantitative proteomics experiments in plants. Trends Plant Sci 17:102 112

84. Benschop JJ, Mohammed S, Oaherty M, Heck AJ, Slijper M, Menke FL (2007) Quantitative phosphoproteomics of early elicitor signaling in Arabidopsis . Mol Cell Proteomics 6:1198 1214

85. Keinath NF, Kierszniowska S, Lorek J, Bourdais G, Kessler SA, Asano H et al (2010) PAMP-induced changes in plasma membrane compartmentalization reveal novel components of plant immunity. J Biol Chem JBC M110:160531

86. Stanislas T, Bouyssie D, Rossignol M, Vesa S, Fromentin J, Morel J et al (2009) Quantitative proteomics reveals a dynamic association of proteins to detergent-resistant membranes upon elicitor signaling in tobacco. Mol Cell Proteomics 8:2186 2198

87. Hebeler R, Oeljeklaus S, Reidegeld KA, Eisenacher M, Stephan C, Sitek B et al (2008) Study of early leaf senescence in Arabidopsis thaliana by quantitative proteomics using reciprocal 14N/15N labeling and difference gel electrophoresis. Mol Cell Proteomics 7:108120

26

M. Mirzaei et al.

89.

Liu H, Sadygov RG, Yates JR (2004) A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem 76:41934201

90.

Podwojski K, Eisenacher M, Kohl M, Turewicz M, Meyer HE, Rahnenf ührer J et al (2010) Peek a peak: a glance at statistics for quantitative label-free proteomics. Expert Rev Proteomics 7:249 261

91.

Zhu W, Smith JW, Huang C-M (2009) Mass spectrometry-based label-free quantitative proteomics. BioMed Res Int 2010:840518

92.

Gammulla CG, Pascovici D, Atwell BJ, Haynes PA (2011) Differential proteomic response of rice ( Oryza sativa ) leaves exposed to high- and low-temperature stress. Proteomics

11:2839

93.

Gammulla CG, Pascovici D, Atwell BJ, Haynes PA (2010) Differential metabolic response of cultured rice (Oryza sativa ) cells exposed to high-and low-temperature stress. Proteomics 10:3001 3019

94.

George IS, Pascovici D, Mirzaei M, Haynes PA (2015) Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism. Proteomics 15:3048 3060

95.

Mirzaei M, Pascovici D, Atwell BJ, Haynes PA (2012) Differential regulation of aquaporins, small GTPases and V-ATPases proteins in rice leaves subjected to drought stress and recovery. Proteomics 12:864 877

96.

Mirzaei M, Soltani N, Sarhadi E, George IS, Neilson KA, Pascovici D et al (2013) Manipulating root water supply elicits major shifts in the shoot proteome. J Proteome Res 13:517 526

97.

Kottapalli KR, Zabet-Moghaddam M, Rowland D, Faircloth W, Mirzaei M, Haynes PA et al (2013) Shotgun label-free quantitative proteomics of water-de cit-stressed midmature peanut (Arachis hypogaea L.) seed. J Proteome Res 12:5048 5057

98.

Uniprot C (2010) The universal protein resource (UniProt) in 2010. Nucleic Acids Res 38:

D142D148

99.

Arnaudo AM, Garcia BA (2013) Proteomic characterization of novel histone post-translational modi cations. Epigenetics Chromatin 6:24

100.

Finkemeier I, Laxa M, Miguet L, Howden AJM, Sweetlove LJ (2011) Proteins of diverse function and subcellular location are lysine acetylated in Arabidopsis . Plant Physiol 155:17791790

101.

Mazzoleni M, Figuet S, Martin-Laffon J, Mininno M, Gilgen A, Leroux M et al (2015) Dual targeting of the protein methyltransferase PrmA contributes to both chloroplastic and mitochondrial ribosomal protein L11 methylation in Arabidopsis . Plant and Cell Physiol:

pcv098

102.

Walton A, Stes E, Cybulski N, Van Bel M, Inigo S, Durand AN et al (2016) It s time for some site -seeing: novel tools to monitor the ubiquitin landscape in Arabidopsis thaliana. Plant Cell: TPC2015-00878-REV

103.

Dam S, Thaysen-Andersen M, Stenkj æ r E, Lorentzen A, Roepstorff P, Packer NH et al (2013) Combined N-glycome and N-glycoproteome analysis of the Lotus japonicus seed globulin fraction shows conservation of protein structure and glycosylation in legumes. J Proteome Res 12:3383 3392

104.

Friso G, Van Wijk KJ (2015) Posttranslational Protein Modi cations in Plant Metabolism. Plant Physiol 169:14691487

105.

Parker CE, Mocanu V, Mocanu M, Dicheva N, Warren MR (2010) Mass spectrometry for post-translational modi cations. In: Alzate O (ed) Neuroproteomics: CRC Press, Boca Raton (FL) Chapter 6

106.

Li J, Silva-Sanchez C, Zhang T, Chen S, Li H (2015) Phosphoproteomics technologies and applications in plant biology research. Front Plant Sci: 6

107.

Silva-Sanchez C, Li H, Chen S (2015) Recent advances and challenges in plant phosphoproteomics. Proteomics 15:1127 1141

1

Applications of Quantitative Proteomics in Plant Research

27

108. Nakagami H, Sugiyama N, Mochida K, Daudi A, Yoshida Y, Toyoda T et al (2010)

Large-scale comparative phosphoproteomics identi es conserved phosphorylation sites in plants. Plant Physiol 153:11611174

109. Hunter T (2007) The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol Cell 28:730 738

110. Vandamme J, Castermans D, Thevelein JM (2012) Molecular mechanisms of feedback inhibition of protein kinase A on intracellular cAMP accumulation. Cell Signal 24:1610

1618

111. Xing T, Ouellet T, Miki BL (2002) Towards genomic and proteomic studies of protein phosphorylation in plantpathogen interactions. Trends Plant Sci 7:224 230

112. Havelund JF, Thelen JJ, Mø ller IM (2015) Biochemistry, proteomics, and phosphoproteomics of plant mitochondria from non-photosynthetic cells. Sub-Cell Proteomics: 98

113. Zargar SM, Nazir M, Rai V, Hajduch M, Agrawal GK, Rakwal R (2015) Towards a common bean proteome atlas: looking at the current state of research and the need for a comprehensive proteome. Front Plant Sci: 6

114. Li G, Boudsocq M, Hem S, Vialaret J, Rossignol M, Maurel C et al (2015) The calcium-dependent protein kinase CPK7 acts on root hydraulic conductivity. Plant Cell Environ 38:13121320

115. Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther TC et al (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325:834 840

116. Choudhary C, Weinert BT, Nishida Y, Verdin E, Mann M (2014) The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol 15:536 550

117. Jeffers V, Sullivan WJ (2012) Lysine acetylation is widespread on proteins of diverse function and localization in the protozoan parasite Toxoplasma gondii . Eukaryot Cell 11:735 742

118. Pandey R, Muller A, Napoli CA, Selinger DA, Pikaard CS, Richards EJ et al (2002) Analysis of histone acetyltransferase and histone deacetylase families of Arabidopsis thaliana suggests functional diversi cation of chromatin modi cation among multicellular eukaryotes. Nucleic Acids Res 30:50365055

119. Hu Y, Qin F, Huang L, Sun Q, Li C, Zhao Y et al (2009) Rice histone deacetylase genes display speci c expression patterns and developmental functions. Biochem Biophys Res Commun 388:266271

120. Servet C, Conde E Silva N, Zhou DX (2010) Histone acetyltransferase AtGCN5/HAG1 is a versatile regulator of developmental and inducible gene expression in Arabidopsis . Mol Plant 3:670677

121. Wang Q, Zhang Y, Yang C, Xiong H, Lin Y, Yao J et al (2010) Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic ux. Science 327:10041007

122. Konig AC, Hartl M, Boersema PJ, Mann M, Finkemeier I (2014) The mitochondrial lysine acetylome of Arabidopsis . Mitochondrion 19 Pt B:252 260

123. Wu X, Oh MH, Schwarz EM, Larue CT, Sivaguru M, Imai BS et al (2011) Lysine acetylation is a widespread protein modi cation for diverse proteins in Arabidopsis . Plant Physiol 155:17691778

124. Smith-Hammond CL, Hoyos E, Miernyk JA (2014) The pea seedling mitochondrial N (epsilon)-lysine acetylome. Mitochondrion 19 Pt B:154 165

125. Smith-Hammond CL, Swatek KN, Johnston ML, Thelen JJ, Miernyk JA (2014) Initial description of the developing soybean seed protein Lys-N(epsilon)-acetylome. J Proteomics 96:5666

126. Melo-Braga MN, Verano-Braga T, Leon IR, Antonacci D, Nogueira FC, Thelen JJ et al (2012) Modulation of protein phosphorylation, N-glycosylation and Lys-acetylation in grape (Vitis vinifera ) mesocarp and exocarp owing to Lobesia botrana infection. Mol Cell Proteomics 11:945 956

28

M. Mirzaei et al.

127. Nallamilli BR, Edelmann MJ, Zhong X, Tan F, Mujahid H, Zhang J et al (2014) Global analysis of lysine acetylation suggests the involvement of protein acetylation in diverse biological processes in rice (Oryza sativa ). PLoS ONE 9:e89283

128. Rardin MJ, Newman JC, Held JM, Cusack MP, Sorensen DJ, Li B et al (2013) Label-free quantitative proteomics of the lysine acetylome in mitochondria identi es substrates of SIRT3 in metabolic pathways. Proc Natl Acad Sci 110:66016606

129. Anderson KA, Hirschey MD (2012) Mitochondrial protein acetylation regulates metabolism. Essays Biochem 52:2335

130. Bedford MT, Richard S (2005) Arginine methylation: an emerging regulatorof protein function. Mol Cell 18:263 272

131. Afjehi-Sadat L, Garcia BA (2013) Comprehending dynamic protein methylation with mass spectrometry. Curr Opin Chem Biol 17:1219

132. Rowland E, Kim J, Bhuiyan NH, Van Wijk KJ (2015) The Arabidopsis Chloroplast stromal N-terminome; complexities of N-terminal protein maturation and stability. Plant Physiol, pp:

01214 02015

133. Vierstra RD (2012) The expanding universe of ubiquitin and ubiquitin-like modi ers. Plant Physiol 160:214

134. Ehrnhoefer DE, Sutton L, Hayden MR (2011) Small changes, big impact: posttranslational modi cations and function of huntingtin in Huntington disease. Neuroscientist:

1073858410390378

135. Devoto A, Muskett PR, Shirasu K (2003) Role of ubiquitination in the regulation of plant defence against pathogens. Curr Opin Plant Biol 6:307 311

136. Moon J, Parry G, Estelle M (2004) The ubiquitin-proteasome pathway and plant development. Plant Cell 16:31813195

137. Sullivan JA, Shirasu K, Deng XW (2003) The diverse roles of ubiquitin and the 26S proteasome in the life of plants. Nat Rev Genet 4:948958

138. Wu X, Gong F, Cao D, Hu X, Wang W (2015) Advances in crop proteomics: PTMs of proteins under abiotic stress. Proteomics 16(5):847 865

139. Vierstra RD (2009) The ubiquitin 26S proteasome system at the nexus of plant biology. Nat Rev Mol Cell Biol 10:385 397

140. Guerra DD, Callis J (2012) Ubiquitin on the move: the ubiquitin modi cation system plays diverse roles in the regulation of endoplasmic reticulum-and plasma membrane-localized proteins. Plant Physiol 160:56 64

141. Kim D-Y, Scalf M, Smith LM, Vierstra RD (2013) Advanced proteomic analyses yield a deep catalog of ubiquitylation targets in Arabidopsis . Plant Cell 25:1523 1540

142. Yu F, Wu Y, Xie Q (2015) Precise protein post-translational modi cations modulate ABI5 activity. Trends Plant Sci 20:569 575

143. Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315:201205

144. Dell A, Morris HR (2001) Glycoprotein structure determination by mass spectrometry. Science 291:23512356

145. Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC (2004) Exploring the O-GlcNAc proteome: direct identi cation of O-GlcNAc-modi ed proteins from the brain. Proc Natl Acad Sci USA 101:13132 13137

146. Albenne C, Canut H, Jamet E (2015) Plant cell wall proteomics: the leadership of Arabidopsis thaliana. Sub-Cell Proteomics: 7

147. Venne AS, Kollipara L, Zahedi RP (2014) The next level of complexity: crosstalk of posttranslational modi cations. Proteomics 14:513 524

148. Champagne A, Boutry M (2013) Proteomics of nonmodel plant species. Proteomics 13:663673

149. Br äutigam A, Shrestha RP, Whitten D, Wilkerson CG, Carr KM, Froehlich JE et al (2008) Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species: comparison of a species-speci c database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes. J Biotechnol 136:44 53

1

Applications of Quantitative Proteomics in Plant Research

29

150. Huang M, Chen T, Chan Z (2006) An evaluation for cross-species proteomics research by publicly available expressed sequence tag database search using tandem mass spectral data. Rapid Commun Mass Spectrom 20:2635 2640

151. Pascovici D, Gardiner DM, Song X, Breen E, Solomon PS, Keighley T et al (2013) Coverage and consistency: bioinformatics aspects of the analysis of multirun iTRAQ experiments with wheat leaves. J Proteome Res 12:4870 4881

152. Mochida K, Shinozaki K (2010) Genomics and bioinformatics resources for crop improvement. Plant Cell Physiol 51:497 523

153. Mochida K, Shinozaki K (2011) Advances in omics and bioinformatics tools for systems analyses of plant functions. Plant Cell Physiol 52:2017 2038

154. Baxevanis Andreas D, Davison Daniel B, Page Roderic DM, Petsko Gregory A, Stein Lincoln D, Stormo Gary D (2003) Current protocols in bioinformatics. Vol. 1, John Wiley & Sons Inc, New York

155. Xie C, Mao X, Huang J, Ding Y, Wu J, Dong S et al (2011) KOBAS 2.0: a web server for annotation and identi cation of enriched pathways and diseases. Nucleic Acids Res 39:

W316W322

156. Du Z, Zhou X, Ling Y, Zhang Z, Su Z (2010) agriGO: a GO analysis toolkit for the agricultural community. Nucleic acids research: W64 70

157. Jensen LJ, Kuhn M, Stark M, Chaffron S, Creevey C, Muller J et al (2009) STRING 8a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res 37:D412 D416

158. Usadel B, Poree F, Nagel A, Lohse M, Czedik-Eysenberg A, Stitt M (2009) A guide to using MapMan to visualize and compare Omics data in plants: a case study in the crop species, Maize . Plant Cell Environ 32:12111229

159. Maere S, Heymans K, Kuiper M (2005) BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 21:3448 3449

160. Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A et al (2009) ClueGO: a cytoscape plug-into decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics 25:1091 1093

161. Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z et al (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:W293 W297

162. Pascovici D, Keighley T, Mirzaei M, Haynes PA, Cooke B (2012) PloGO: plotting gene ontology annotation and abundance in multi-condition proteomics experiments. Proteomics 12:406 410

163. Nakagami H, Sugiyama N, Ishihama Y, Shirasu K (2012) Shotguns in the front line:

phosphoproteomics in plants. Plant Cell Physiol 53:118 124

164. Cox J, Mann M (2011) Quantitative, high-resolution proteomics for data-driven systems biology. Annu Rev Biochem 80:273 299

165. Yao Q, Ge H, Wu S, Zhang N, Chen W, Xu C et al (2014) P3DB 3.0: from plant phosphorylation sites to protein networks. Nucleic Acids Res 42:D1206 D1213

166. Bessarabova M, Ishkin A, Jebailey L, Nikolskaya T, Nikolsky Y (2012) Knowledge-based analysis of proteomics data. BMC Bioinformatics 13:1

Chapter 2

Seed Proteomics: An Overview

Kanika Narula, Arunima Sinha, Toshiba Haider, Niranjan Chakraborty and Subhra Chakraborty

Abstract Seed is vital for propagation of spermatophytes in biome and as food source for inhabitants of the earth. Studies on seed proteins provide platform for new avenues to explore molecular networks and pathways governing seed lling, maturation, germination, and seedling formation. Protein expression changes of three genetically different sub-regions of angiosperm seeds are re ected in ordered chain of biological events represented from family differences in different taxas. Different families of angiosperm show divergence of seed protein evolution and thus provide insights into seed structure and function. A gamut of information is available on seed proteomic datasets from approximately 3500 proteins that impinge on protein function in diverse plant families. The functional modularity of seed proteins were compared amongst species that span from dicot to moncot and diploid to polyploid. Transitions of protein complement revealed difference between dormancy and germination towards understanding biological check point at translational level. Goal of this chapter is to critically review data available till date on seed proteomic studies and identify family and cross genera knowledge gaps. The information thus obtained would unravel new components and an unparallel understanding of the molecular processes underlying translational and post-translational variations under different conditions that involves histodifferen- tiation and organogenesis of the seed.

Keywords Crop plant Seed proteomics Seed Dormancy Germination

Kanika Narula and Arunima Sinha are equally contributed in this work.

K. Narula A. Sinha T. Haider N. Chakraborty S. Chakraborty (&) National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India e-mail: subhrac@hotmail.com

32

K. Narula et al.

2.1 Introduction

Evolution of angiosperm has facilitated the transitional dominance of seed producing plants into a terrestrial environment. According to the fossil records their rst appearance dated back to 365 million years ago when majority of landscape was successfully and naturally selected for angiosperm survival [ 1 ]. The physiological and genetic control of seed is in part responsible for dispersal mechanism which proved to be a spectacular phenomenon responsible for prevalence of seed plants on livable planet, the earth. True seed is fertilized mature ovule possessing an embryonic plant, stored food, and a protective coat. A major factor during angiosperm embryogenesis is the switch from the radial symmetry of the globular embryo to the bilateral symmetry followed by differentiation of cotyledons and embryonal axis [ 2 ]. Seed development and germination is a continuous and ne-tuned process with natural circumscription engrossing three phases of embryogenesis recognized as rapid cell division, deposi- tion of reserves, and desiccation. Commonly, mature seeds are classied as albu- minous and ex-albuminous depending on the presence or absence of endosperm. Earlier in 1946, Martin studied the internal morphology of seeds belonging to 1287 genera of angiosperm and classi ed them based on size of the embryo in relation to endosperm and differences in the size, shape, and position of embryo in the seed. The developmental program of monocot and dicot embryogenesis is different but a highly ordered phenomenon. In addition, regulatory pathways, dissection of complex traits, and developmental reprogramming re ects the fundamental differences of the molecular biology of two taxas. To build new perspectives approach for the physiological and biochemical factors controlling seed traits in two taxas, omics studies of seed development were per- formed since year 2000 exploiting the availability of genome sequence and related resources [3 ]. Further, advancement in high-throughput proteome analysis using gel and non-gel based approaches provided detailed protein pro le at different develop- mental stages which might help to elucidate the regulatory network of embryogenesis related proteins. Currently, proteomics is playing an important role in: (i) under- standing plant biology, (ii) developing plant biomarkers for human health and food security and (iii) food analysis and bio-safety issues [ 4 ]. Seed proteomics research has largely focused on agriculturally important crop plants. Typically, these seeds are obtained from a commercial source. It is reasonable to assume that they are genetically uniform thus it is not biased by the contributions of a contaminating proteome. The strategy of using combinatorial-ligand random peptide beads appears to have sub- stantial potential to deplete the supra-abundant SSP from input samples [ 5 ]. In addition, proteomic approaches also dissected double fertilization reprogramming at translational and post-translational level in two different clades of angiosperm. This review aims to compile proteomic analyses performed until now to understand evo- lution of monocot and dicot seed protein patterning, analyzing protein pro le of inaccessible regions of seeds, regulatory networks of underlying mechanism of embryogenesis and lling to modulate and outline the strategies to identify candidate seed proteins controlling the regulatory switches.

2

Seed Proteomics: An Overview

33

2.2 Perspect of Seed Biology

Seed is a multifaceted organ which develops from fertilized ovule and is important for plant survival, evolution and agricultural production. Strictly de ned, seed development is accompanied with many distinct metabolic, cellular and physio- logical changes including imbibition, respiration, RNA and protein synthesis, enzyme activities within its surviving structures like endosperm, nucellus, cotyle- don, teguments and components including funiculus and integument. Seed biology research has been conducted extensively due to its importance in food industry. Seeds of different genera and family of angiosperm have diverse importance being used for oil, spices, bre, carbohydrate, fat, secondary metabolites and in brewing industry. They ensure the perpetuation of life forms and spread of the species to new areas by means of autochory, anemochory, hydrochory, and zoochory [ 6 ]. Distinct physio-chemical properties of diverse angiosperm seeds compelled the researchers to study their molecular entities including protein complement to understand cellular circuitry correlated to the morphological and physiological adaptations from germination to seedling growth.

2.3 Protein Organization of Seed Parts

In angiosperm, the female gametophyte is seated deep in the ovarian cavity far away from stigma where pollen germinates. There is sufcient evidence to suggest that gametophyte speci c proteins were present during fertilization phase [ 7 ]. Further, the triploid endosperm, the most common nutritive tissue for the devel- oping seed in angiosperm, maintains a critical protein balance with embryo and maternal tissues. Nuclear, helobial and cellular endosperm shows either symmetric or asymmetric growth that is exempli ed by differences in protein patterns [ 8 ]. He et al. [ 9 ] performed comparative proteomic analysis of wheat embryo and endo- sperm during seed germination. The most abundant proteins both in the embryo and endosperm were found to be seed storage proteins such as legumins, vicilins and albumins. Housekeeping enzymes, actin-binding pro lin, defense-related protein kinases, nonspeci c lipid transfer protein and proteins involved in general meta- bolism were also identi ed. In monocot, morphologically and biochemically dis- tinct outermost layer of endosperm namely aleurone exhibit differences in protein organization and constitutes an important accumulatory reserve tissue. Following a predetermined mode of development, fertilized egg give rise to embryo which show differences in morphology, anatomy and biochemistry both in monocot and dicots, and protein composition is no exception. Proteomic studies had begun to reveal proteins that are necessary for the events such as pattern formation, cell differen- tiation and organ development. Up till now approximately 1500 diverse proteins are reported to be active in dicot plants and 928 proteins in monocot plants (Table 2.1 ). Many of these are expressed in speci c cell types and regions of seeds. For modular

34

K. Narula et al.

Table 2.1 A comprehensive list of angiosperm seed proteome study

Clade

Family

Plant

Organ/tissue

References a

Dicot

Brassicaceae

Arabidopsis

Whole seed

[25]

     

Whole seed

[23]

     

Whole seed

[19]

     

Whole seed

[21]

     

Whole seed

[27]

     

Whole seed

[26]

     

Whole seed

[22]

     

Whole seed

[17]

     

Whole seed

[18]

   

Camelina

Whole seed

[48]

   

Castor bean

Nucellus

[45]

     

Whole seed

[44]

     

Whole seed

[42]

     

Whole seed

[46]

   

Mustard

Whole seed

[30]

     

Whole seed

[49]

   

Oilseed rape

Embryo

[35]

   

Rapeseed

Cotyledon

[31]

     

Endosperm

[34]

     

Whole seed

[28]

     

Whole seed

[33]

     

Whole seed

[32]

     

Whole seed

[47]

     

Whole seed

[29]

   

Rapeseed,

Whole seed

[43]

arabidopsis

 

Euphorbiaceae

Jatropha

Embryo and endosperm

[36]

     

Embryo, endosperm

[37]

     

Endosperm

[41]

     

Inner integument

[39]

     

Whole seed

[40]

     

Whole seed

[38]

 

Leguminoceae

Chickpea

Whole seed

[80]

   

Common

Whole seed

[

78]

bean

 
   

Lentil

Whole seed

[79]

     

Cotyledons, hypocotyl

[73]

     

Endosperm,

[74]

     

Whole seed

[71]

     

Whole seed

[72]

(continued)

2

Seed Proteomics: An Overview

35

Table 2.1 (continued)

Clade

Family

Plant

Organ/tissue

References a

   

Medicago

Whole seed

[68]

     

Whole seed

[66]

     

Whole seed

[69]

   

Medicago,

Whole seed

[70]

black bean

   

Mungbean

Whole seed

[81]

   

Pea

Embryonic axis

[57]

     

Whole seed

[75]

     

Whole seed

[76]

   

Peanut

Whole seed

[82]

     

Whole seed and testa

[83]

   

Pigeon pea

Whole seed

[84]

   

Soybean

Cotyledon

[64]

     

Cotyledon

[63]

     

Hypocotyl, radicle

[65]

     

Seed coat

[13]

     

Seed coat

[12]

     

Whole seed

[51]

     

Whole seed

[52]

     

Whole seed

[5 ]

     

Whole seed

[60]

     

Whole seed

[61]

     

Whole seed

[58]

     

Whole seed

[62]

     

Whole seed

[59]

     

Whole seed

[57]

     

Whole seed

[56]

     

Whole seed

[55]

     

Whole seed

[54]

     

Whole seed

[53]

     

Whole seed

[59]

 

Amaranthceae

Sugarbeet

Whole seed

[85]

 

Anacardiaceae

Cashew

Cotyledon

[86]

 

Cucurbitaceae

Melon

Whole seed

[89]

 

Oleaceae

Olive

Whole seed

[90]

 

Rosaceae

Cherry

Cotyledons, embryos, testae

[88]

 

Rubiaceae

Coffee

Embryo

[92]

 

Solanaceae

Tomato

Embryo, endosperm

[87]

 

Theaceae

Tea

Whole seed

[93]

 

Vitaceae

Grape

Endosperm

[91]

(continued)

36

K. Narula et al.

Table 2.1 (continued)

Clade

Family

Plant

Organ/tissue

References a

Monocot

Poaceae

Barley

Aleurone

[150]

     

Aleurone, endosperm, embryo, whole Seed

[144]

     

Endosperm

[143]

     

Whole seed

[154]

     

Whole seed

[147]

     

Whole seed

[142]

     

Whole seed

[145]

     

Whole seed

[148]

     

Whole seed

[149]

     

Whole seed

[146]

     

Whole seed

[152]

     

Whole seed

[151]

     

Whole seed

[153]

   

Maize

Embryo

[122]

     

Embryo

[135]

     

Embryo

[134]

     

Embryo

[133]

     

Embryo

[132]

     

Embryo, endosperm

[139]

     

Embryo, endosperm

[140]

     

Embryo, endosperm

[102]

     

Endosperm

[138]

     

Scutellum

[136]

     

Whole seed

[137]

   

Rice

Embryo

[114]

     

Embryo

[115]

     

Embryo